The aldehyde dehydrogenase 2 rs671 variant enhances amyloid β pathology

In the ALDH2 rs671 variant, a guanine changes to an adenine, resulting in a dramatic decrease in the catalytic activity of the enzyme. Population-based data are contradictory about whether this variant increases the risk of Alzheimer’s disease. In East Asian populations, the prevalence of the ALDH2 rs671 variant is 30–50%, making the National Human Brain Bank for Development and Function (the largest brain bank in East Asia) an important resource to explore the link between the ALDH2 rs671 polymorphism and Alzheimer’s disease pathology. Here, using 469 postmortem brains, we find that while the ALDH2 rs671 variant is associated with increased plaque deposits and a higher Aβ40/42 ratio, it is not an independent risk factor for Alzheimer’s disease. Mechanistically, we show that lower ALDH2 activity leads to 4-HNE accumulation in the brain. The (R)−4-HNE enantiomer adducts to residue Lys53 of C99, favoring Aβ40 generation in the Golgi apparatus. Decreased ALDH2 activity also lowers inflammatory factor secretion, as well as amyloid β phagocytosis and spread in brains of patients with Alzheimer’s disease. We thus define the relationship between the ALDH2 rs671 polymorphism and amyloid β pathology, and find that ALDH2 rs671 is a key regulator of Aβ40 or Aβ42 generation.

In the legend of Fig. 6, state what siNC; it is explained only in Fig. 7. Fig. 1: indicates that 6E10 is an anti A beta plaque antibody.P.22 'both disrupted the opposing effects' should be 'disrupted both opposing effects'.
Discussion of Alda-1 on Page 24: The authors speculate why the clinical trial by ALDEA was terminated based on an unreviewed comment on the web.In fact, ALDEA used another alda (not Alda-1) in a small phase 1 clinical study.The study was completed and not terminated for toxicity.Rather, the work was terminated because the inventors realized that the clinical trial size needed to be bigger than they initially thought.They pulled out the rest of the investment and folded ALDEA.Since then, the IP was licensed to Foresee, and a clinical trial for Alda in a pediatric indication, Fanconi Anemia, is ongoing (NCT04522375) after a successful safety study was completed.
The use of Alda-1 in this report: How Alda-1 or daidzein was dosed in the mice is not provided in the Method section.Perhaps the noted toxicity was due to a single intraperitoneal injection of a very high drug dose or the vehicle used (also not indicated in the manuscript).
In published studies, Alda-1 was dosed in WT mice for several months and was found to be safe, for example, in models of post-myocardial infarction heart failure, in a model of Parkinson's disease, and in a chronic model of ethanol-induced neurotoxicity.In all these studies, Alda-1 was delivered at 10mg/kd/day using a slow delivery via a subcutaneous Alzet pump.
Reviewer #2 (Remarks to the Author): The manuscript titled "Aldehyde dehydrogenase 2 rs671 variant enhances Alzheimer's disease pathology" by Wang et al describes a comprehensive genetic, in vitro and in vivo study to link the rs671 A variant to Alzheimer's disease (AD), but not as a genetic risk factor, but instead as a modifier variant resulting in increased amyloid beta pathology identified in post-mortem brain tissue.A major strength of this study is the large East Asian ancestry cohort used to directly compare genotype with neuropathology.
There have been multiple recent studies, particularly in East Asian populations, investigating the link between rs671 genotype and AD, including meta-analyses of these studies (example: doi.org/10.1186/s13643-022-02050-y). Wang et al have not sought to replicate these studies, but instead investigating the potential biological relevance of this genetic variant in relation to AD.In vitro and in vivo studies determine a functional reduction/inhibition of ALDH2 enzyme (in A variant carriers -GA/AA genotypes) resulting in impaired microglia action leading to aggregation through the increased spreading of AB plaques.The paper is well-written, and the data is mostly well presented.

Major comments:
1.A large number of studies have been conducted investigating the ALDH2 rs671 variant in multiple diseases such as listed in the following publication: doi.org/10.1186/s13643-022-02050-y.It would be important for the authors to mention that how the rs671 variant has been identified as a risk factor and/or susceptibility locus in multiple diseases, not just in relation to AD.
2. With such a large cohort to select from, as a reviewer it is frustrating to see a study design not matched for sex and age, especially since the authors conducted such a comprehensive and timeconsuming IHC of 8 regions per patient selected.The GG and GA genotype cohorts selected for IHC both do not have 1:1 F:M ratio selected (the AA genotype cohort is 1:1 F:M matched).I also note that the average age for IHC selected samples is GG: 81.5 years, GA: 83 years and AA: ./%-+P>:JK% 7P <HG<>JG OBLA LA>BJ KLM=P =>KB@G BG LA:L# BG @>G>J:E# 0S =>IHKBLBHG BG<J>:K>K OBLA :@># :G= LA> 541 :G= 36580 <HAHJLK <AHK>G A:N> BG<J>:KBG@ :@> KAHOBG@ BG<J>:KBG@ 0S I:LAHEH@P% Can the authors please include an additional table or information to demonstrate that the samples selected were representative of the genotype cohort.For example, what is the average age/stdev of AA genotype AD patients compared to the 4 selected for IHC and ELISA.Can the authors please also clarify why the cohorts were not matched for sex or age.
3. Since the authors have made a point in their study design not to include heavy drinkers, it would be nice to see a brief discussion point (maybe 2 sentences) about the role of ALDH2 rs671 in alcohol metabolism and AD, since this is a key published function of the rs671 variant, particularly in East Asian populations.
4. As mentioned above, a strength to this study is using pathology-diagnosed AD (vs cliniciandiagnosed AD).If the data is available, co-pathologies would be interesting to include in Supplementary Table 1 and whether there is any correlation with rs671 genotype.
5. There is a contradictory statement on page 25 in the Discussion "However, there are not enough AA individuals to demonstrated the association with AD pathology change", yet in multiple other locations throughout the text including in the title of the manuscript, the authors describe that the Q0624) JK,-( N:JB:GL IHKBLBN>EP <HJJ>E:L>K OBLA 0S I:LAHEH@PR% 6. Do East Asian populations generally have elevated AB pathology compared to non-East Asian populations?If so, would the increased prevalence of the A allele account for this? 7. Given the increasing genotype-phenotype correlations that are being identified through large biobank efforts, this is a topical analysis.Can the authors please include a discussion point on what is the clinical relevance of this genotype-phenotype correlation.Could it be useful for subgrouping for clinical trials or post-hoc analysis of clinical trial efficacy?8.There is no Table legend for supplementary figure 1 (4-page pdf) provided with manuscript submission.What is the column "with other CNS disorders"?What other CNS disorders were investigated during post-mortem pathology.9.It is not clear to me the value of including RNA-sequencing data since there are only 2/50 included participants with the AA genotype, and none overlap the samples included in this study.Can additional AA genotype patients undergo RNA-seq? 10.Can the author please comment on why "the effect of ALDH2 rs671 polymorphism should be IJHI>JEP :=CMKL>= OA>G J:GDBG@ LA> 0S I:LAHEH@P K<HJ> HG IHKLFHJL>F ;J:BGKR% 9ABK O:K GHL <E>:J to me.
Minor comments: 11.Please include HUGO nomenclature for any genetic variant at first mention in the manuscript (nucleotide and protein change) and glossary, including accession number.
12. Please consider using the word "sex" instead of "gender" throughout the text/figures/tables if you are referring to the patient's sex chromosome genotype (i.e., XX or XY).
The authors have in a screen of 329 human brain samples of investigated the role of ALDH2 rs671 polymorphism in AD.This genetic variant has previously been shown to present a risk for hypertension, diabetes and coronary heart disease in the Asian population.For AD, the impact of this polymorphism have so far been contradictory.The investigation was initiated on a population biobank consisting of 59% male and 41% female samples, on which genotyping was conducted.In turn, amyloid plaque assessment was done to find potential correlation between alleles and phenotype.Further, detailed analysis on Ab levels was done with ELISA.Next, the authors investigated the effects of the Aldh2-gene on Ab-peptide ratios in the APPSwe mouse model of AD.This was done both with a knock-out and by pharmacological modulation of the Aldh2.The author then investigate the potential accumulation of 4-HNE on Ab-peptide ratios.Adduction of 4-HNE to Ab-peptides are found with mass spectrometry, and phenotypic effects were observed with an in vivo assay using HEK293 cells.Overall the manuscript present many interesting findings and connect a large screening study with detailed analysis of molecular mechanisms that highlight the importance of Aldh2 in AD.However, I have major concerns regarding the proteomic data that the authors must address.
I am confused by how the authors present their proteomic data with volcano plots in extended data figs 6 and 14.It is not clear from the figures how many replicates were measured with proteomics.There are apparently no statistics involved in determining whether a protein has been up-or downregulated.Is extended data figure 6 based on only a single replicate of each condition?Rather, the authors choose an arbitrary cutoff at abs(foldchange)>1.25 in extended data figure 6 and abs(foldchange)>1.17 in extended data figure 14.This is not an acceptable method to show significant effects in differential expression, because a protein with only logFC=0.5 can have adj.p.value > 0.0001, but something logFC=3 can have adj.p.value=0.5.Instead, the authors must reprocess the data with proper statistical methods, such as Student's t-test or suitable moderated t-tests using either the R-packages limma or DESeq2.The appropriate statistical analysis must be done, including multiple testing correction such as the Benjamini-Hochberg procedure, where volcano plots should show logFC on the x-axis and log10(-adj.p.value) on the yaxis.
Data availability: The proteomic data must be submitted to PRIDE (public repository).This has not been done.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): The manuscript of Wang et al. describes exciting new data on the role of ALDH2 variant (rs671), common in the East Asian population, in Alzheimer's disease.The study does not only explain the potentially conflicting data on the association of this variant with the disease in humans but also provides molecular and cellular basis for how ALDH2 reduced function causes it.This comprehensive study relies on a relatively large collection of brain autopsy samples, multiple cell culture models, two mouse models, and several pharmacological tools.
In short, the study demonstrated that diminished clearance of the aldehyde 4HNE due to reduced ALDH2 activity increases the modification of the A-beta precursor protein, thus altering gamma secretase activity which results in the accumulation of oligomerized A beta 40.The study shows that the modified Abeta aggregates accumulate at the Golgi and that in microglia, this causes increased proinflammatory effect.
An exciting analysis of the microglia behavior (using cultured BV2 and histochemistry of human and animal models of AD) indicates that decreased ALDH2 activity impairs microglial Abeta phagocytosis, leading to increased diffusion of toxic Abeta throughout the brain.Finally, the authors identified the differential effect of the racemic 4HNE and the potential molecular explanation for its effect on A beta40 accumulation.All in all, the study covers many aspects -perhaps even too many.Nevertheless, it is easy to follow and written clearly, and the data support their conclusions.

A few comments:
Data analysis: It is not clear how quantitation of the histochemistry data was conducted.Also, how was potential bias in the analysis prevented (e.g. was the analysis conducted by a reviewer blinded to the experimental conditions?).
For 4-HNE staining, average IOD with at least 3 randomly selected fields was calculated in each slide.
The quantification of IHC staining was conducted in a blinded fashion by the author (Y.C.) who was blinded to the experimental groups using Image Pro Plus software.
We added the information in the Methods 'Tissue processing for immunohistochemistry' section.Also, the immunofluorescence data show a single cell.A lower magnification containing more cells should be provided in the supplementary material and the number (%) of cells showing the phenotype should be indicated.

Response:
To verify the results in Fig. 6a, we reperformed the immunofluorescent staining on WT 293T and (R)-4-HNE treated 293T at a final concentration of 2 µM (R)-4-HNE for 24h.After double staining with anti-C1/6.1 (staining for APP) and anti-RCAS1 (staining for Golgi), lower magnification images containing more cells were added in the Supplementary Fig. 11a.The statistical analysis of APP + RCAS1 + cells were indicated in this figure .To verify the results in Fig 6i , we first prepared VPS35 knock down and control 293T cells using siVPS35 and relative siNC.Then the negative control (siNC-193T) and VPS35 knock down (siVPS35-293T) cells were treated with PBS or 2 µM of (R)-4-HNE for 24h.Double staining for APP (anti-C1/6.1) and Golgi (anti-RCAS1) were conducted.Lower magnification images and statistical analysis of APP + RCAS1 + cells were provided in the Supplementary Fig. 11f.
The revised Supplementary Fig. 11a,f  In the summary, introduction, and results, the authors describe the opposing functions of microglia as a double edge sword, providing explanation to the meaning of that only in the discussion.It would be better to refer to these findings as opposing effects on microglial functions and use the expression 'double edge sword' once in the discussion.
Response: Thanks for your kind suggestion.We now provide more clear expression using opposing effects of microglia and replaced the expression "double edge sword" in the Introduction and Results

sections.
Abstract -'clearly' should be removed from the last sentence.
Response: We removed "clearly" from the last sentence in the Abstract.The sentence was corrected to "We thus defined the relationship between ALDH2 rs671 polymorphism and AD, and found ALDH2 rs671 Results -The study did not 'enroll' the participants to the study.Rather, the author used existing autopsy samples from the Brain Bank.The language should be corrected accordingly.

Response:
In the Results section, we removed the 'enrolled'.The sentence was now revised to '…a total of 329 participants from the Human Brain Bank were included for ALDH2 rs671 sequencing,…'.
In several places, the authors use 'a decrease of ALDH2 levels', the 'decreased ALDH2 levels", etc.It should be 'lower ALDH2 protein levels' Response: To provide a clear description, we revised the 'a decrease of ALDH2 levels', 'a decrease of ALDH2 activities, and 'the decreased ALDH2 activities' to be 'lower ALDH2 protein levels', or 'lower ALDH2 activities' in several places throughout the whole manuscript.On p. 8, upper paragraph, the authors discuss ADH1B activity.However, only levels of the enzyme were assessed.Also, Daidzein inhibits (not suppressed) ALDH2 enzyme activity.
Response: ADH1B is the first enzyme in alcohol metabolism, the upstream gene of ALDH2, that catalyzes alcohols into aldehydes.
ALDH2 is a key mitochondrial aldehyde dehydrogenase, which catalyze acetaldehyde to acetic acid.ALDH2 rs671 G>A (Glu487Lys) leads to a substantial decrease in dehydrogenase activity.According to previous reports, we used 2 methods to reduce the enzyme activity of ALDH2 in SH-SY5Y and N2a-APPswe cells: RNA silencing with siALDH2, or dardzin treatment.Daidzin is a commonly used ALDH2 antagonist which specifically inhibited ALDH2 enzyme activity.We then detected/verified the lower/reduced ALDH2 enzymatic activity using the colorimetric Mitochondrial Aldehyde Dehydrogenase (ALDH2) Activity Assay Kit (Abcam, ab115348, Cambridge, MA).And we verified decrease of ALDH2 activity in SH-SY5Y and N2a-APPswe cells in both methods.The results were provided in Fig. 2g and Supplementary Fig. 5i,m Supplementary Fig. 5i, m.N2a-APPswe cells with Aldh2 knockdown (i) and with daidzin treatment (60 i@# NWZ ,0 P "U#& Enzymatic activity of Aldh2 in cell lysates, measured over 120 min.
In the legend of Fig. 6, state what siNC; it is explained only in Fig. 7.
Response: siNC is the negative control small interfering RNA, randomly scrambled sequences that do not target any genes.In this study, the siNC sequence is shown in Methods "Small interfering RNA transfection" section.we used the same sequence as siNC in human RNA silencing and mouse RNA silencing.We added "siNC, negative control small interfering RNA" in Fig. 2   Response: GM XZW^QLML \PM QVLQKI\QWV e.9)( IV\QJWLa "IV\Q%h%IUaTWQL )%).IV\QJWLa#f QV :QO& )J TMOMVL& P.22 'both disrupted the opposing effects' should be 'disrupted both opposing effects'.

Response:
We revised the sentence to "This study demonstrated that reduced ALDH2 activity disrupted both opposing effects of microglia" in the last paragraph in P22.
Discussion of Alda-1 on Page 24: The authors speculate why the clinical trial by ALDEA was terminated based on an unreviewed comment on the web.In fact, ALDEA used another alda (not Alda-1) in a small phase 1 clinical study.The study was completed and not terminated for toxicity.Rather, the work was terminated because the inventors realized that the clinical trial size needed to be bigger than they initially thought.They pulled out the rest of the investment and folded ALDEA.Since then, the IP was licensed to Foresee, and a clinical trial for Alda in a pediatric indication, Fanconi Anemia, is ongoing (NCT04522375) after a successful safety study was completed.
The use of Alda-1 in this report: How Alda-1 or daidzein was dosed in the mice is not provided in the Method section.Perhaps the noted toxicity was due to a single intraperitoneal injection of a very high drug dose or the vehicle used (also not indicated in the manuscript).
In published studies, Alda-1 was dosed in WT mice for several months and was found to be safe, for example, in models of post-myocardial infarction heart failure, in a model of Parkinson's disease, and in a chronic model of ethanol-induced neurotoxicity.In all these studies, Alda-1 was delivered at 10mg/kd/day using a slow delivery via a subcutaneous Alzet pump.
Response: Thanks for your detail introduction and suggestion.
In this study, the dose of Alda-1 used APP/PS1 mice is 15 mg/kg/day of body weight, via intragastric infusion (iG) for two months.In the Methods "Animal experimental models and drug treatment" section, we added the missing details and clearly stated the administration of Alda-1 and daidzin in mice, including the reagents used for dissolution, the dosing and procedures of administration.This added description was as shown below: Drug treatment and brain tissue preparation.Alda-1 was dissolved in 50% DMSO/50% PEG-400 (v/v).
The dissolving strategy used for Alda-1 and Daidzin in this study was according to the manufacture instructions.We noted that our dissolving vehicle for Alda-1 is consistent to the previous report.
In proteomics analysis part, we reorganized the screening of differentially expressed proteins induced by Daidzin or Alda-1 in mouse neuron N2a-APPswe and in mouse microglia BV2 cells.According to previous published papers, we set adjusted P < 0.05 and protein expression fold change <0.83 or >1.20 as filtering criteria.We found that daidzin treatment mainly affected fatty acid metabolism in N2a-APPswe (Fig. 3a).Alda-1 did not significantly alter proteins profiling in N2a-APPswe (Supplementary Fig. 14c).
In BV2, Alda-1 treatment altered expression of proteins related to metabolisms of lipid or steroids (Supplementary Fig. 14f-h).After bioinformatic analysis, we did not find significant alteration of apoptosis related proteins.So, we revised the description of Alda-1 in Discussion section.
In this section, we added the discussion of Alda-1 in APP/PS1 mice reported by Joshi et al.  14a-b).In vitro, Alda-1 did not significantly affect the wide-type neuron with no significant altering of protein expression profiling in N2a-APPswe (Supplementary Fig. 14c).
Wide-type microglia BV2 showed inhibited proliferation (Supplementary Fig. 14d-e) but unaffected phagocytic ability (Supplementary Fig. 12h) after treatment of 20 µM or more Alda-1.The altered metabolisms of lipid or steroids (Supplementary Fig. 14f-h, Supplementary Table 8) may account for the inhibition effect induced by Alda-1.
Reviewer #2 (Remarks to the Author): The manuscript titled "Aldehyde dehydrogenase 2 rs671 variant enhances Alzheimer's disease pathology" by Wang et al describes a comprehensive genetic, in vitro and in vivo study to link the rs671 A variant to Alzheimer's disease (AD), but not as a genetic risk factor, but instead as a modifier variant resulting in increased amyloid beta pathology identified in post-mortem brain tissue.A major strength of this study is the large East Asian ancestry cohort used to directly compare genotype with neuropathology.
There have been multiple recent studies, particularly in East Asian populations, investigating the link between rs671 genotype and AD, including meta-analyses of these studies (example: doi.org/10.1186/s13643-022-02050-y). Wang et al have not sought to replicate these studies, but instead investigating the potential biological relevance of this genetic variant in relation to AD.In vitro and in vivo studies determine a functional reduction/inhibition of ALDH2 enzyme (in A variant carriers -GA/AA genotypes) resulting in impaired microglia action leading to aggregation through the increased spreading of AB plaques.The paper is well-written, and the data is mostly well presented.
Major comments: 1.A large number of studies have been conducted investigating the ALDH2 rs671 variant in multiple diseases such as listed in the following publication: doi.org/10.1186/s13643-022-02050-y.It would be important for the authors to mention that how the rs671 variant has been identified as a risk factor and/or susceptibility locus in multiple diseases, not just in relation to AD.
Response: Thank you for these critical comments.Indeed, rs671 mutation was linked to multiple diseases, suggesting it leads to a significant change of ALDH2 and the importance of ALDH2 in many disease related pathways.We added the association between ALDH2 rs671 polymorphism and multiple diseases in the Introduction section.The added sentence is as follows: A large number of studies demonstrated increased association between ALDH2 rs671 polymorphism and many diseases 4,5 , including elevated risk of cancers 6 and cerebral vascular disease 7 after alcohol consumption, opposite effects in varied cardiovascular diseases 3 , in ALDH2 rs671 A-allele carriers.
2. With such a large cohort to select from, as a reviewer it is frustrating to see a study design not matched for sex and age, especially since the authors conducted such a comprehensive and time-consuming IHC of 8 regions per patient selected.The GG and GA genotype cohorts selected for IHC both do not have 1:1 F:M ratio selected (the AA genotype cohort is 1:1 F:M matched).I also note that the average age for IHC selected samples is GG: 81.5 years, GA: 83 years and AA: 89.75 years.My concern with their study design Can the authors please include an additional table or information to demonstrate that the samples selected were representative of the genotype cohort.For example, what is the average age/stdev of AA genotype AD patients compared to the 4 selected for IHC and ELISA.Can the authors please also clarify why the cohorts were not matched for sex or age.R1.1).

Response
We also calculated the average age and sex distribution in GG, GA, AA genotype groups of the total 469 individuals included in the study.The results were shown in the Table R1  AA-allele brains than GG brains after chronic alcohol consumption in mice 41 .
4. As mentioned above, a strength to this study is using pathology-diagnosed AD (vs clinician-diagnosed AD).If the data is available, co-pathologies would be interesting to include in Supplementary Table 1 and whether there is any correlation with rs671 genotype.

Response:
We agree with the reviewer's opinion about the correlation of co-pathologies with rs671 polymorphism.
While our study is not a prospective cohort study.We used the human brain tissues which were already preserved in the human brain bank.These donors did not receive cognitive function tests before death.

Fig. 1 :
Fig. 1: indicates that 6E10 is an anti A beta plaque antibody.

Response:
Thanks for your suggestion.Alcohol consumption is a risk factor for health.While in rs671 GA/AA individuals, the lower activity of dehydrogenase ALDH2 would drastically slow down the metabolism of alcohol-derived acetaldehyde, which is more toxic for cells with the active aldehyde group in the molecule.So, alcohol consumption aggravates the aldehyde load in rs671 A carriers.We searched several published papers and added their results and discussions that associated with alcohol consumption and AD in the second paragraph of Discussion section.The added discussion is as follows: ?W_ KWVKMV\ZI\QWV WN M\PIVWT [PW_[ XZW\MK\Q^M MNNMK\[ IOIQV[\ 5h \W`QKQ\a QV PQXXWKIUXIT VM]ZWV[ 39 and cardiac-cerebral vascular disease in GG genotype individuals 7,40 .Excessive ethanol exposure is detrimental to the brains and is a higher risk factor for AD 39 .ALDH2 rs671 G>A greatly reduces alcohol metabolism inducing toxic aldehyde load.Joshi et al. demonstrated aggravated neuropathology in ALDH2 JI[ML WV I NW]Z%XWQV\ [KITM$ (%,& 7WOVQ\Q^MTa VWZUIT _I[ LMNQVML I[ 97WO c)&(3 UQTL KWOVQ\Q^M QUXIQZUMV\ as ECog 1.0-2.0;and dementia as ECog >2.0[2].In our study, we obtained the average Ecog scores of 303 donors and analyzed the correlation between rs671 gene polymorphism and the average Ecog score.The average Ecog score of each individual was provided in Supplementary

Table 4 .
What is NC? 18.A small formatting comment for supplementary table that goes across 4 pages (Supplementary table 1?), please put headings on each page and table legend on pdf.
.1 below.Tabel R1.1 Distribution of sex and age of individuals included in this study.Since the authors have made a point in their study design not to include heavy drinkers, it would be nice to see a brief discussion point (maybe 2 sentences) about the role of ALDH2 rs671 in alcohol metabolism and AD, since this is a key published function of the rs671 variant, particularly in East Asian populations.

Table 1 .
Though the ordinal logistic regression analysis showed no higher risk of rs671 polymorphism on the Ecog score (Fig 1a), we found an increased proportion of individuals with average Ecog score >2 in GA/AA genotype populations.The results were shown in Table R1.2 below.Table R1.2.The distribution of average ECog score in 303 postmortem brain donors.